Tissue-Specific KO of ECM Proteins

  • Emilio Hirsch
  • Mara Brancaccio
  • Fiorella Altruda
Part of the Methods in Molecular Biology™ book series (MIMB, volume 139)

Abstract

The analysis of phenotypes caused by null and mutant alleles is a very powerful means to understand gene function in vivo. Historically, this experimental approach has been widely and successfully used in invertebrate models. Now, thanks to the gene-targeting technology in ES cells, the genome of a mammalian organism such as the mouse can be artificially modified by precise alterations. The system exploits the ability of ES cells to be cultured and manipulated in vitro without losing their totipotency (1,2). Mutations in specific genes can be achieved by in vitro selection of ES cell clones in which the locus of interest has been targeted by homologous recombination (3,4). The peculiar property of being totipotent allows ES cells, once injected in the cavity of a blastocyst, to contribute to the formation of all cell types of a chimeric embryo. Whenever a chimeric mouse possesses ES-derived germ cells, the mutation can be propagated to its offspring. Heterozygous mice are then mated to generate the homozygous offspring needed for phenotypic analysis.

Keywords

Cellulose DMSO Cage Agarose Chloroform 

References

  1. 1.
    Evans, M. J. and Kaufman, M. H. (1981) Establishment in culture of pluripotent cells from mouse embryos. Nature 292, 154–156.PubMedCrossRefGoogle Scholar
  2. 2.
    Martin, G. (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma cells. Proc. Natl. Acad. Sci. USA 78, 7634–7638.PubMedCrossRefGoogle Scholar
  3. 3.
    Smithies, O., Gergg, R. G., Boggs, S. S., Koralewski, M. A., and Kuckerlapati, M. S. (1985) Insertion of DNA sequences into the human chromosomal β-globin locus by homologous recombination. Nature 317, 230–234.PubMedCrossRefGoogle Scholar
  4. 4.
    Thomas, K. R. and Capecchi, M. R. (1987) Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51, 503–512.PubMedCrossRefGoogle Scholar
  5. 5.
    Gu, H., Marth, J. D., Orban, P. C., Mossmann, H., and Rajewsky, K. (1994). Deletion of a DNA polymerase beta gene segment in T cells using cell type-spe-cific gene targeting. Science 265, 103–106.PubMedCrossRefGoogle Scholar
  6. 6.
    Baudoin, C., Goumans, M. J., Mummery, C., and Sonnenberg, A. (1998) Knockout and knockin of the beta1 exon D define distinct roles for integrin splice variants in heart function and embryonic development. Genes Dev. 12, 1202–1216.PubMedCrossRefGoogle Scholar
  7. 7.
    Nagy, A., Moens, C., Ivanyi, E., Pawling, J., Gertsenstein, M., Hadjantonakis, A. K., et al. (1998) Dissecting the role of N-myc in development using a single targeting vector to generate a series of alleles. Curr. Biol. 8, 661–664.PubMedCrossRefGoogle Scholar
  8. 8.
    Sternberg, N. and Hoess, R. (1983). The molecular genetics of bacteriophage P1. Annu. Rev. Genet. 17, 123–154.PubMedCrossRefGoogle Scholar
  9. 9.
    Kilby, N. J., Snaith, M. R., and Murray, J. A. (1993) Site-specific recombinases: tools for genome engineering. Trends Genet. 9, 413–421.PubMedCrossRefGoogle Scholar
  10. 10.
    Kuhn, R., Schwenk, F., Aguet, M., and Rajewsky, K. (1995). Inducible gene tar-geting in mice. Science 269, 1427–1429.PubMedCrossRefGoogle Scholar
  11. 11.
    Rajewsky, K., Gu, H., Kuhn, R., Betz, U. A., Muller, W., Roes, J., and Schwenk, F. (1996) Conditional gene targeting. J. Clin. Invest. 98, 600–603.PubMedCrossRefGoogle Scholar
  12. 12.
    Akagi, K. Sandig, V., Vooijs, M., Van der Valk, M., Giovannini, M., Strauss, M., and Berns, A. (1997) Cre-mediated somatic site-specific recombination in mice. Nucleic Acids Res. 25, 1766–1773.PubMedCrossRefGoogle Scholar
  13. 13.
    Betz, U. A., Vosshenrich, C. A., Rajewsky, K., and Muller, W. (1996) Bypass of lethality with mosaic mice generated by Cre-loxP-mediated recombination. Curr. Biol. 6, 1307–1316.PubMedCrossRefGoogle Scholar
  14. 14.
    Aszodi, A., Pfeifer, A., Wendel, M., Hiripi, L., and Fässler, R. (1998) Mouse models for extracellular matrix diseases. J. Mol. Med. 76, 238–252.PubMedCrossRefGoogle Scholar
  15. 15.
    Morrison-Graham, K., and Weston, J. A. (1989) Mouse mutants provide new insights into the role of extracellular matrix in cell migration and differentiation. Trends Genet. 5, 116–121.PubMedCrossRefGoogle Scholar
  16. 16.
    Fässler, R., Schnegelsberg, P. N., Dausman, J., Shinya, T., Muragaki, Y., McCarthy, M. T., et al. (1994) Mice lacking alpha 1 (IX) collagen develop noninflammatory degenerative joint disease. Proc. Natl. Acad. Sci. USA 91, 5070–5074.PubMedCrossRefGoogle Scholar
  17. 17.
    Mundlos, S. and Olsen, B. R. (1997) Heritable diseases of the skeleton. Part II: molecular insights into skeletal development-matrix components and their homeostasis. FASEB J. 11, 227–233.PubMedGoogle Scholar
  18. 18.
    Bruckner-Tuderman, L. and Bruckner, P. (1998) Genetic diseases of the extracellular matrix: more than just connective tissue disorders. J. Mol. Med. 76, 226–237.PubMedCrossRefGoogle Scholar
  19. 19.
    Gilmour, D. T., Lyon, G. J., Carlton, M. B., Sanes, J. R., Cunningham, J. M., Anderson, J. R., et al. (1998) Mice deficient for the secreted glycoprotein SPARC/ osteonectin/BM40 develop normally but show severe age-onset cataract forma-tion and disruption of the lens. EMBO J. 17, 1860–1870.PubMedCrossRefGoogle Scholar
  20. 20.
    Lawler, J., Sunday, M., Thibert, V., Duquette, M., George, E. L., Rayburn, H., and Hynes R. O. (1998) Thrombospondin-1 is required for normal murine pulmo-nary homeostasis and its absence causes pneumonia. J. Clin. Invest. 101, 982–992.PubMedCrossRefGoogle Scholar
  21. 21.
    Saga, Y., Yagi, T., Ikawa, Y., Sakakura, T., and Aizawa, S. Mice develop normally without tenascin. Genes Dev. 6, 1821–1831.Google Scholar
  22. 22.
    Forsberg, E., Hirsch, E., Frohlich, L., Meyer, M., Ekblom, P., Aszodi, A., et al. (1996) Skin wounds and severed nerves heal normally in mice lacking tenascin C. Proc. Natl. Acad. Sci. USA 93, 6594–6599.PubMedCrossRefGoogle Scholar
  23. 23.
    Erickson, H. P. (1993) Gene knockouts of c-src, transforming growth factor beta 1, and tenascin suggest superfluous, nonfunctional expression of proteins. J. CellBiol. 120, 1079–1081.CrossRefGoogle Scholar
  24. 24.
    Tybulewicz, V. L., Crawford, C. E., Jackson, P. K., Bronson, R. T., and Mulligan, R. C. (1991). Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. Cell 65, 1153–1163.PubMedCrossRefGoogle Scholar
  25. 25.
    te Riele, H., Maandag, E. R., and Berns, A. (1992) Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. Proc. Natl. Acad. Sci. USA 89, 5128–5132.CrossRefGoogle Scholar
  26. 26.
    Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., and Roder, J. C. (1993) Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA 90, 8424–8428.PubMedCrossRefGoogle Scholar
  27. 27.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Second Edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  28. 28.
    Hasty, P., Rivera-Perez, J., and Bradley, A. (1991) The length of homology required for gene targeting in embryonic stem cells. Mol. CellBiol. 11, 5586–5591.Google Scholar
  29. 29.
    Zhang, H., Hasty, P., and Bradley, A. (1994). Targeting frequency for deletion vectors in embryonic stem cells. Mol. Cell. Biol. 14, 2404–2410.PubMedGoogle Scholar
  30. 30.
    Reichardt, H. M., Kaestner, K. H., Tuckermann, J., Kretz, O., Wessely, O., Bock, R., et al. (1998). DNA binding of the glucocorticoid receptor is not essential for survival. Cell 93, 531–541.PubMedCrossRefGoogle Scholar
  31. 31.
    Li, Z. W., Stark, G., Gotz, J., Rulicke, T., Gschwind, M., Huber, G., et al. (1996) Generation of mice with a 200-kb amyloid precursor protein gene deletion by Cre recombinase-mediated site-specific recombination in embryonic stem cells. Proc. Natl. Acad. Sci. USA 93, 6158–6162.PubMedCrossRefGoogle Scholar
  32. 32.
    Braun, R. E., Lo, D., Pinkert, C. A., Widera, G., Flavell, R. A., Palmiter, R. D., and Brinster, R. L. (1990) Infertility in male transgenic mice: disruption of sperm development by HSV-tk expression in postmeiotic germ cells. Biol. Reprod. 43, 684–963.PubMedCrossRefGoogle Scholar
  33. 33.
    Hogan, B., Beddington, R., Costantini, F., and Lacy, E. (1994) Manipulating the Mouse Embryo. A Laboratory Manual. Second Edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  34. 34.
    Hirsch, E., Iglesias, A., Potocnik, A. J., Hartmann, U., and Fassler, R. (1996) Impaired migration but not differentiation of haematopoietic stem cells in the absence of beta1 integrins. Nature 380, 171–175.PubMedCrossRefGoogle Scholar
  35. 35.
    Mortenssen, R. M., Zubiaur M., Neer, E. J., and Seidman, J. G. (1991) Embryonic stem cells lacking a functional inhibitory G-protein subunit (ai2) produced by gene targeting of both alleles. Proc. Natl. Acad. Sci. USA 88, 7036–7040.CrossRefGoogle Scholar
  36. 36.
    Sasaki, T., Forsberg, E., Bloch, W., Addicks, K., Fässler, R., and Timpl, R. (1998) Deficiency of beta 1 integrins in teratoma interferes with basement membrane assembly and laminin-1 expression. Exp. Cell Res. 238, 70–81.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2000

Authors and Affiliations

  • Emilio Hirsch
    • 1
  • Mara Brancaccio
    • 2
  • Fiorella Altruda
    • 2
  1. 1.Universita di TorinoTorino
  2. 2.Universita di TorinoTorinoItaly

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